A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae. Issue 6 (13th March 2018)

• Record Type: Journal Article
• Authors: Vickery, Christopher R.; Cardenas, Javier; Bowman, Marianne E.; Burkart, Michael D.; Da Silva, Nancy A.; Noel, Joseph P.
• Is Part Of: Biotechnology and bioengineering. Volume 115:Issue 6(2018)
• Page Start: 1394
• Page End: 1402
• Publication Date: 2018-03-13
• Subjects: 2‐pyrone synthase -- enzyme engineering -- polyketide -- Saccharomyces cerevisiae -- triacetic acid lactone; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.26564
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high‐value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2‐pyrone synthase (2‐PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae . Initial in vitro characterization of 2‐PS led to the identification of active site variants with improved kinetic properties relative to wildtype. Further in vivo evaluation in S. cerevisiae suggested certain 2‐PS mutations altered enzyme stability during fermentation. In vivo experiments also revealed beneficial cysteine to serine mutations that were not initially explored due to their distance from the active site of 2‐PS, leading to the design of additional 2‐PS enzymes. While these variants showed varying catalytic efficiencies in vitro, they exhibited up to 2.5‐fold increases in TAL production when expressed in S. cerevisiae . Coupling of the 2‐PS variant [C35S, C372S] to an engineered S. cerevisiae strain led to over 10 g/L TAL at 38% of theoretical yield following fed‐batch fermentation, the highest reported to date. Our studies demonstrate the success of a coupled in vitro/in vivo approach to engineering enzymes and provide insight on cysteine‐rich enzymes and design principles toward their use in non‐native microbial hosts. Abstract : A coupled in vitro/in vivo approach for engineering the plant enzyme 2‐PS was implemented to increase the synthesis of triacetic acid lactone (TAL), an important chemical precursor, in the yeast Saccharomyces cerevisiae . Simultaneous engineering of the yeast host and manipulation of the enzyme via site‐directed mutagenesis resulted in a yeast strain capable of producing greater than 10 g/L TAL. This is the highest production of TAL achieved in S. cerevisiae to date, and marks an important step towards understanding the interplay between hosts and enzyme activity with respect to the bioproduction of valuable chemical compounds.